curacy of the photometric analyzer also makes it useful for quantitative measurements of ultraviolet absorbing substances. Curve I in Figure 8 shows the chromatogram obtained with the recording unit when 0.25 mg. of three nucleotides were run through a small Dowex 1 formate coluntn with gradient 0 -+ lilf formic acid. The absorption values of Curve I1 were obtained as a result of a planimetric calculation based on the recorded Curve I. R'ith the knowledge of the positions of the tubes in the fraction collector and their place on the strip chart, the area between the base line and the Curve I for cach single tube
was determined. The values obtained, which represent the amounts of nucleotide fractions contained in the tubes, were plotted as Curve 11. Finally the content of nucleotide in the tubes was directly determined in the Beckman DU spectrophotometer. Table I shows that there is reasonably close agreement between the absorption values calculated and the values read in the Beckman DU spectrophotometer. ACKNOWLEDGMENT
Skillful assistance of Nils-Olof Hedlund and Thore Persson is gratefully acknowledged.
LITERATURE CITED
(1) Agren, G., Acta Chem. Scand. 14,2065 (1960). (2)67.55 Agren, (1962). G., Acta SOC. Mea. Upsal. (3) Igren, G.,' Engstrom, L., Acta Chem. Scand. 11,1087 (1958). (4) Engstrom, L., Ibid., 63, 128 (1958). (5) Lindahl, P. E., Cancer Research 20, 841 (1960). (6) Sjoquist, J., Rydberg, C.-E., Svensson, R., Kgl. Fysiogruf. Sallskap. Lund, F&h. 26, No. 13 (1956). ( 7 ) Verdier, C.-H. de, Acta SOC.Med.
Upsal. 57,393 (1952). RECEIVEDfor review Nay 6, 1963.
Accepted December 16, 1963. The investigation WAS supported by grants from the Ther6se and Johan Andersson Foundation and the Swedish Cancer Society.
A Quantitative Chromatographic Determination of Cysteic Acid in Amino Acid Mixtures on Ion Exchange Papers JAN HARTEL' and A. J. G. PLEUMEEKERS2 Central laboratory TNO, Delft, Netherlands
b- A
new and rapid method for determining cysteic acid in a mixture o f amino acids has been cleveloped. For the separation, use i s made o f chemically modified paper containing quaternary ammonium groups. Quantitative determination can b e done on the paper densitometr cally as well as spectrophotometrically (after elution). The densitometric determinotion i s rapid but not very exact. The spectrophotometric method gives more reproducible values with a relative standard deviation of 3%.
xETHoDs for the determination of cysteic acid in amino acid mixtures rely on the fact that this amino acid is more acidic than others. Therefore, methods are usually based on ion exchange chromatography and paper electrophoresis. Ion eschsnge column (hromatography, described by Schrani, Moore, and Bigwood (6) gives escellent separations, but the method is rather laborious and time consuming. TT'ith high voltage paper electrophoresis, Zuber, Ziegler, and Zahn (8) obtained good results in much shorter time. Diehl ($) and Bauters, Lefebvrc, and Van Overbeke (1) modified the electrophoresis conditions and wed lorn voltage paper electrophoresis to give a simpler and less expensive method. OST
Present address, De Wit's Textile Nijverheid Yj. V., Helmrind, Xetherlands. Present address, Fibre Research Institute TNO, Delft, Netherlands.
To further simplify the method, we used ion exchange papers developed in the Central Laboratory, TNO. These papers, made by chemical modification, will be produced by Macherey and Nagel in Duren, Germany, and will be available with strongly or weakly acid or base groups. They have proved to be very useful in separating mixtures of compounds with ionic groups, especially amino acids ( 5 ) . For the determination of cysteic acid, we used a paper with strongly basic quaternary ammonium groups. After separation, the cysteic acid spots could be determined by densitometry or, after elution, by spectrophotometry, EXPERIMENTAL
Apparatus and Reagents. RIacherey and Sage1 ion exchange paper with strongly basic groups; Agla micrometer syringe; Photovolt densitometer, Model 501-A; Eppendorf colorimeter; and, for part of the experiments, Bausch and Lomb Spectronic 20. The ninhydrin reagent is a 0.5% solution in 96% ethanol and contains 0.2y0glacial acetic acid. To prepare the copper reagent (?), 10 ml. of saturated copper nitrate solution is mixed with 0.2 ml. of concentrated nitric acid and made up to 1 liter with acetone. Procedure. The ion exchange paper, available in the chloride form, is conditioned by washing i t overnight with the mobile phase, 0.34M chloroacetic acid. Excess chloroacetic acid is removed by rinsing the paper three times with distilled water.
After drying for 20 minutes a t 65' C., the paper can be stored until needed. On a 25-em. long sheet of paper, a starting line is drawn 3 cm. from the lower edge. The cysteic acid solutions are applied with a micrometer syringe a t 2.5-em. intervals along this line. I n general, a maximum quantity of 5 pl. is applied. For larger quantities, spotting is repeated with intermittent drying of the spot. I n addition to the unknowns, three different quantities of a standard solution of cysteic acid (0.1% in water) are applied t o the same chromatogram. All applications are in duplicate. One place on the starting line is left open for the determination of the paper blank. The quantity of cysteic acid applied varies between 0.4 and 1.0 bg. for the densitometric determination and between 2 and 20 pg. for the spectrophotometric determination. The paper is made into a cylinder by connecting the side edges with a cotton thread. This paper roll is placed in a glass cylinder which can be closed. The chromatogram is developed with 0.34.V chloroacetic acid by the ascending technique. When the liquid has progressed 15 em. from the starting line (40 to 60 minutea), the chromatogram is removed and dried a t 65" C. for 20 minutes. The paper is pulled through the ninhydrin solution from the starting line to the liquid front and is dried hanging vertically with the starting line up. These precautions prevent migration of heavily concentrated spots. After impregnation, the paper is dried a t 65" C. for 20 minutes. Quantitative. DENSITOMETRIC DETERMINATION. The colored chromatogram is cut into strips according VOL. 36,
NO. 6. MAY 1964
1021
paper blank. The concentration of cysteic acid is determined by reference to a prepared standard curve. RESULTS AND DISCUSSION
Cysteic acid in wool was readily separated from the normal amino acids, see Figure 1. To find out if cysteic acid could also be separated from other compounds containing a sulfonic group, 4rg. 8rg. lPr9. 6rl. 4rl. a chromatogram v a s made of cysteic Standard Curve Wool Hydrolyzate acid, cysteic-S-sulfonic acid, threonine Figure 1. Chromatogram of cysteic sulfate, and serine sulfate. (Courtesy acid and a wool hydrolyzate to be K. Ziegler, Deutsches Wollforschungused for the spectrophotometric desinstitut an der Technischen Hochschule termination of cysteic acid in wool Aachen.) Sulfonic-containing compounds may be present as hydrolyzates in chemically treated wool. Asshown to the spot locations. The spots are in Figure 2, only serine sulfate was not measured on a densitometer with a separated from cysteic acid under these slit of 6 X 1 mm. or a round aperture conditions. Other experiments showed of 2 mm., and a filter of 570 mp. With that serine sulfate and cysteic acid have the slit, the total spot is measured and the area described by the extincthe same RI value in this system. tion curve is taken as a measure for Reproducibility. I n both methodsthe concentration of the cysteic acid. densitometric as well as spectrophotoWith the round aperture, the maximum metric-the standard curves are not density of the spot is determined. I n fully reproducible. Therefore, sf andthe first case, the standard curve is ard curves have t o be prepared with obtained by plotting the values for each chromatogram. standard against their concentration The standards may be applied from in the second case, the values are one solution by using different quantities plotted against the logarithm of the or a constant quantity of solutions of concentration, Block ( 2 ) . SPECTROPHOTONETRIC DETERMINA- varying concentrations may be applied. TION. For the spectrophotometric Since the quantities applied are very measurement, the colored spots are small, especially for the densitometric treated with copper solution to make determination, the method using soluelution possible. tions of varying concentrations is most The front spots are cut off the strips sensitive as regards variations in spot and the remaining chromatogram is dimensions. dipped in freshly prepared copper soluThe densitometric determination is tion. After 15 minutes, the paper strip more rapid but less accurate than the is removed and dried for 30 minutes in the dark a t room temperature. All spectrophotometric method. manipulations after the treatment with In the spectrophotometric method, copper solution have to be carried out the standard curve is a straight line in the dark, even the coloring itself. for amounts up to 20 pg. of cysteic acid. These paper strips are eluted with To establish the reproducibility of methanol by the method described by the spectrophotometric method, a series Dent (3). The elutions are performed of 16 determinations was carried out in a closed glass tank. For rapid with the hydrolyzate of a bleached wool elution, it may be useful to deposit some sample. The hydrolyzate was prepared methanol on the bottom of the tank. The spots are eluted with about 1 ml. by boiling 1 gram of wool in 10 ml. of of methanol in 45 to 60 minutes. The hydrochloric acid (1:l) for 4 hours. solutions are collected and made up to The acid was evaporated in vacuo mark with methanol in 5-ml. volumetric under nitrogen and the residue was disffasks. Spectrophotometric measuresolved in 10 ml. of water. Because of ments are made a t 504 mp against a the displacing action of the chloride ion, large amounts of hydrochloric acid should not remain in the solution. The data shown in Table I were obtained Table 1. Reproducibility of Spectrofrom four chromatograms, each conphotometric Determination of Cysteic taining four spots of the hydrolyzate Acid Content of a Bleached,Wool (6 p l . ) and four spots for the standard Sample in Wt./Wt. % curve (two, 8 pg. and two, 12 pg). Chromatogram 1 2 3 4 spot No. ACKNOWLEDGMENT 1 2 3 4
1.24 1.25 1.20 1.25
Mean value Std. dev. 1022
1 . 2 2 1.29 1.24 1.32 1 . 2 3 1.31 1.24 1.24 1.25 0.3%
ANALYTICAL CHEMISTRY
1.18 1.26 1.24 1.24
The authors thank G. J. Schuringa, C. van’,Bochove, and E. A. Leidelmeyer for valuable discussions and J. Wolvekamp and M. A. Andriessen for their technical assistance. They also thank K. Ziegler for samples which he supplied.
Figure 2. Separation of mixtures containing cysteic acid and some other acids 1. Cysteic acid 11. Cysteic acid and cysteic-S-sulfonic acid
Ill. Cysteic acid and threonine sulfate IV. Cysteic acid and serine sulfate V. Mtxture of I-IV
LITERATURE CITED
(1) Bauters, M., Lefebvre, L., Overbeke, M. Van, Bull. I. T. F 80, 7 (1959). (2) Block, R. Y., “A Manual of Paper
Chromatography and Paper Electrophoresis,” 2nd ed., Academic Press, New York, 1958. (3) Dent, C. E., Biochern. J . 41, 240 (1947). (4) Diehl, J. F., ANAL. CHEM.31, 1204 (19*59\. \ - - - - , -
(5) Hartel, J., Copper, J. A. S., Bochove C. Van, Rec. Trav. Chim. 82, 264 (1963). . ( 6 ) Schram, E., Moore, S., Bigwood, E. J., Baochem. J . 57, 33 (1954). (7) Wieland, T., Kaverav, E., Nature 168, 77 (1951). (8) Zuber, H., Ziegler, K., Zahn, H., Naturforsch. 126,531 (1957).
RECEIVEDfor review August 27, 1962. Resubmitted September 20, 1963. Accepted December 11, 1963.
Correction Fluorescent Compounds for Calibration of Excitation and Emission Units of Spect rofI uor0 meter I n this article by R. J. Argauer and C. E. White [ANAL. CHEX 36, 368 (1964)] on page 369, colun~n1, lines 44 and 45, should read “Seven and a half grams (0.05 mole) of p-dimethylaminobenzaldehyde, 9.0 grams (0.05 mole) of p-nitrophenylacetic acid, , .”
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